About 20% of U.S. population have congenitally missing teeth. Chance of tooth loss also increases along with a person's age. A naturally regenerated or in vitro generated implantable tooth would be a perfect replacement in such situations. Tissue engineering using the self-renewing stem cells is becoming one of the most powerfull and promising approaches to repair or replace an injured tissue or organ. Assembly of a functional organ from directed differentiating stem cells will have significant clinical application. Our long-term effort is to make human tooth regeneration become a reality. We envision two approaches to regenerate the human tooth in the future: (1) identifying key factor(s) that can initiate tooth developmental program, and applying the factor(s) to allow the rebuilding of teeth; and (2) generating implantable teeth in vitro from stem cells. In this application, we will use rodents as a model system test the possibility of generating an implantable tooth organ from bone marrow stromal cells (BMSCs). Three specific aims are proposed. Aim 1 is proposed to examine the properties of the potential odontogenic competence in the rat BMSCs. We will first confirm our preliminary results that BMSCs could differentiate into odontoblasts when an odontogenic microenvironment was encountered. Whether BMSCs can respond to odontogenic signals in a similar way as dental mesenchyme, in terms of induction of gene expression and tooth bud formation, will be tested by tissue recombination and protein-soaked bead implantation. Additionally, if BMP4 can confer BMSCs odontogenic competence will also be tested. Aim 2 will determine if BMSCs can be induced or "conditioned" by an odontogenic microenvironment or potential odontogenic signaling molecules, and to acquire odontogenic potential, capable of inducing tooth formation when recombined with an embryonic epithelium, or to become odontogenic competent, capable of responding to odontogenic signals to support tooth formation. In vitro tissue recombination, organ culture, in situ hybridization, and kidney capsule grafting will be employed to address these questions. Lastly in Aim 3, we will test if in vitro assembled tooth germ is implantable and can develop into a functional tooth in the oral cavity by exo uteru surgery. Information obtained from these studies can be translated into application in humans in the near future.